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First published online 27 June 2006
doi: 10.1242/jcs.03013

Journal of Cell Science 119, 2975-2984 (2006)
Published by The Company of Biologists 2006
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Thyroid hormone receptors TR{alpha}1 and TRß differentially regulate gene expression of Kcnq4 and prestin during final differentiation of outer hair cells

Harald Winter1, Claudia Braig1, Ulrike Zimmermann1, Hyun-Soon Geisler1, Jürgen-Theodor Fränzer1, Thomas Weber1, Matthias Ley1, Jutta Engel2, Martina Knirsch2, Karl Bauer3, Stephanie Christ3, Edward J. Walsh4, JoAnn McGee4, Iris Köpschall1, Karin Rohbock1 and Marlies Knipper1,*

1 University of Tübingen, Department of Otolaryngology, Tübingen Hearing Research Centre (THRC), Laboratory of Molecular Neurobiology, Elfriede-Aulhorn-Str. 5, 72076 Tübingen, Germany
2 University of Tübingen, Institute of Physiology II and Department of Otolaryngology, THRC, Gmelinstr. 5, 72076 Tübingen, Germany
3 Max-Planck-Institute for Experimental Endocrinology, Feodor-Lynen-Str. 7, 30625 Hannover, Germany
4 Developmental Auditory Physiology Laboratory, Boys Town National Research Hospital, 555 North 30th Street, Omaha, NE 68131, USA


Figure 1
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  		Fig. 1. Coincident redistribution of KCNQ4 and prestin in rat OHCs. (A) Immunohistochemistry shows KCNQ4 (green) and prestin (red) in OHCs before (P8) and at onset of hearing (P12). (B) Double immunohistochemistry of prestin (red) and KCNQ4 (green) in a mature (P21) OHC. Arrows, KCNQ4 and prestin protein; arrowheads, basal pole of OHCs. Bars, 20 µm.

 

Figure 2
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  		Fig. 2. KCNQ4 expression in control and hypothyroid rats. (A) Double immunohistochemistry of prestin (red) and KCNQ4 (green) in OHCs of control and hypothyroid (hypo) rats at P21. (B) Using northern blotting, both, the ~3.9 kb and ~3.8 kb Kcnq4 transcripts (filled arrows), are reduced in the cochleae of P12 hypothyroid rats (hypo) compared with controls (con), detected with a Kcnq4-specific antisense riboprobe (as). Sense control shows no signal. Blots were probed with a cyclophilin-antisense probe (open arrow) as a housekeeping gene. (C) Reduced Kcnq4 mRNA levels in the cochlea of P12 hypothyroid rats (hypo) compared with controls (con) is also noted using semi-quantitative RT-PCR. The housekeeping gene cyclophilin was used as a control. (D) Localization of Kcnq4 mRNA in control and hypothyroid (hypo) rat cochleae at P18 using in situ hybridization. Note the strong signals in OHCs and a weaker signal in IHC of controls, whereas no signals are detectable in hair cells of hypothyroid rats. Bars, 10 µm (A); 20 µm (D).

 

Figure 3
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  		Fig. 3. KCNQ4 and prestin expression in OHCs of TR{alpha}1–/–ß–/– mutants compared with hypothyroidism at P10. (A,B) In OHCs of hypothyroid rats (hypo), prestin is expressed but its immature distribution persists (A, red) whereas KCNQ4 expression is absent (B, red). (C,D) In TR{alpha}1–/–/ß–/– mutants, both prestin (C, red) and KCNQ4 (D, red) are expressed but their immature distribution persists. Double immunohistochemistry was performed with synaptophysin (green). Open arrowheads, basal pole of OHCs; filled arrowheads, KCNQ4 and prestin protein. Bar, 20 µm.

 

Figure 4
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  		Fig. 4. Deletion of TR{alpha}1 but not TRß exclusively restores KCNQ4 expression in OHCs despite hypothyroidism. (A,C) At P12, in hypothyroid TRß–/– mutants (hypo), KCNQ4 protein was not observed (A), prestin was expressed but its distribution remained immature (C). (B,D) In hypothyroid TR{alpha}1–/– mutants (hypo), KCNQ4 appears normal (B), whereas prestin persists in an immature distribution (D). (E,F) T4-mediated rescue leads to a normal adult expression of KCNQ4 in hypothyroid TRß–/– mutants (E), like in T4-treated TR{alpha}1–/– mutants (F). (G,H) T4 does not rescue prestin from its immature pattern in hypothyroid TRß–/– mutants (G), but does so in hypothyroid TR{alpha}1–/– mutants (H). Sections were coimmunolabeled with synaptophysin (green) and DAPI (blue). Arrows, basal pole of OHC. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. Binding of TR to TREKCNQ4 analyzed by EMSA. (A) Sequence and localization of TRE-1KCNQ4, TRE-2KCNQ4, and TRE-3KCNQ4 relative to the transcriptional start site. Hexamers are underlined and nucleotides matching consensus sequence are in bold. (B) EMSA of the three TREKCNQ4. Recombinant chicken TR{alpha} (cTR{alpha}) shifts [32P]TRE-3 (lane 4), but not [32P]TRE-1 (lane 2) or [32P]TRE-2 (lane 3) to positions similar to [32P]DR4 (lane 1), which correspond to a higher cTR{alpha} homodimer and a lower cTR{alpha} monomer complex. In competitor experiments, the interaction between cTR{alpha} and [32P]TRE-3 (lane 4) is significantly reduced in the presence of an excess of unlabeled competitor oligomer TRE3KCNQ4 (lane 5) and DR4 (lane 6) but not in the presence of unlabeled TFIID-specific oligomer (lane 7). Mutations in the 5' half of unlabeled TRE-3KCNQ4 (TRE-3mut1) have no influence on the capability of cTR{alpha} to shift the [32P]TRE-3 (lane 8). Mutations in the 3' half of TRE-3KCNQ4 (TRE-3mut2) still abolish the interaction (lane 9) to a similar degree as unlabeled TRE-3KCNQ4 or DR4 competitor oligomers (lanes 5, 6). (C) Sequence annotation of the mutated TRE-3KCNQ4 oligomers used as competitors. Mutated residues are highlighted. (D) Functional analysis of the putative human KCNQ4 promoter and the TRE-3KCNQ4 in reporter gene assays. Introduction of the human KCNQ4 promoter into a promoterless vector leads to 4.3-fold induction of reporter gene expression compared with the promoterless vector (4.3-fold ±0.3 s.d.; n=4; white column). Insertion of the TRE-3KCNQ4 upstream of the human KCNQ4 promoter leads to a decreased 2.1-fold induction of reporter gene expression (2.1±0.5 s.d., ***P<0.05; n=4) in the absence of ligands (black column) which could be overcome upon addition of 150 nM T3 and 1.5 µM ATRA restoring promoter activity to 4.1-fold induction of reporter gene expression (4.1±1.1 s.d., ***P<0.05; n=3; gray column). (E) Compared with control conditions (lane 1) addition of 600 nM T3 (lane 2) impairs the binding of cTR{alpha} homodimers (higher shift band) to [32P]TRE3KCNQ4 whereas monomer binding is unaffected.

 

Figure 6
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  		Fig. 6. Comparison of binding properties of TRE-3KCNQ4 and TREPrest by EMSA. In vitro translated rat TR{alpha} (rTR{alpha}) and rTRß shifts [32P]TRE-3KCNQ4 (lanes 1,3) as well as [32P]TREPrest (lane 5,7) to two complexes with different electromobility (filled and open arrowheads). The interaction of rTR{alpha} or rTRß with [32P]TRE3KCNQ4 is significantly reduced in the presence of an excess of unlabeled TRE-3KCNQ4 competitor oligomers (lanes 2,4). The same is true for rTR{alpha} or rTRß with [32P]TREPrest by using TREPrest as a competitor (lanes 6,8).

 

Figure 7
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  		Fig. 7. KCNQ4 and prestin expression in OHCs of heterozygous TR{alpha}1m/+ mutants at P13. (A,B) In OHCs of TR{alpha}1+/+ wild type, expression and distribution of KCNQ4 (A, red) and prestin (B, red) is normal. (C,D) In OHCs of TR{alpha}1m/+ mutants KCNQ4 is completely absent (C) whereas prestin expression and distribution is normal (D). (E,F) By contrast, KCNQ4 expression in vestibular hair cells (VHC) is normal within the same sections of both the wild type (E) and TR{alpha}1m/+ mutants (F). Sections were co-immunolabeled with synaptophysin (green) and DAPI (blue). Bar, 20 µm.

 

Figure 8
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  		Fig. 8. Model of the regulation of prestin and Kcnq4 gene expression by an interplay of TH and distinct TR isoforms in a single OHC. (A) In the absence of TH, apo-TRß exerts only a weak repression of prestin, allowing basal transcription. (B) Transcription is activated in the presence of TH by TRß. (C) In the absence of TH, apo-TR{alpha}1 represses Kcnq4. (D) In the presence of TH, TR{alpha}1 activates Kcnq4. Putative co-repressors (CoR) and co-activators (CoA) and transient local peak of cochlear D2 activity may be involved. D2 activity is depicted according to data from Campos-Barros et al. (Campos-Barros et al., 2000Go). D2, 5'-deiodinase type 2; PIC, pre-initiation complex.

 

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© The Company of Biologists Ltd 2006